Identification Of Fat And Lean Phenotypes In Chickens Using Molecular Markers

ABSTRACT

The present invention provides methods of screening chickens to determine those more likely to have a lean or fat phenotype. The invention also provides methods of screening chickens to identify a polymorphism associated with a fat or lean phenotype.

This application claims the benefit of provisional application Ser. No.60/530,051 filed Dec. 16, 2003, which is hereby incorporated byreference.

REFERENCE TO U.S. GOVERNMENT SUPPORT

This work is supported by a grant from the USDA-IFAFS, Animal GenomeProgram (Award Number 00-52100-9614). The United States government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to method for identifying the phenotype ofa chicken using a genetic polymorphism associated with a fat or leanphenotype. More particularly the invention relates to method ofidentifying a fat or lean chicken phenotype by determining the presenceof an insertion/deletion associated with a fat or lean phenotype in oneor both of the duplicated chicken Spot 14 genes, also referred to asthyroid hormone responsive Spot 14 protein (THRSP α and THRSP β)paralogs.

BACKGROUND OF THE INVENTION

Over the last decades intensive selection on growth rate has been donein broiler chicken strains developed for meat production. However,fatness has also been increased, leading to excessive adiposity. Byreducing feed efficiency and lean meat yield, this excess of fat tissueis a major drawback for production.

In order to decipher the metabolic and genetic mechanisms involved inthe regulation of fatness in the chicken, some investigators havedeveloped experimental models of adiposity. Lean and fat chicken lineshave been divergently selected from adipose tissue weight (Leclerq etal., 1980) and for very low density lipoprotein (VLDL) plasmaconcentration (Whitehead, C. C., Griffin, H. D., 1984). Studiesperformed in lean and fat lines developed by Leclercq et al (1980)indicate that the difference in adiposity between lines was not theresult of a difference in food consumption or in nutrient utilization.Stearoyl-Co-A desaturase activity and plasma VLDL concentration werefound to be higher in the fat line (Legrand, P. and Hermier, D., 1992),suggesting a higher lipogenesis rate in this line.

In the chicken, lipogenesis occurs essentially in the liver, the adiposetissue being only a storage tissue (O'Hea, E. K. and Leveille, G. A.,1968; Griffin et al., 1992).

The Spot 14 gene, also referred to as thyroid hormone responsive Spot 14protein (THRSP), encodes a small acidic protein that was discovered inearlier studies of thyroid hormone action on hepatocytes (Seelig et al.,1981; Jump et al., 1984; Liaw and Towle, 1984). Although the exactmolecular mechanism is not clear, THRSP is strongly implicated as atranscription factor that controls expression of major lipogenicenzymes. For instance, THRSP is only expressed in lipogenic tissue suchas liver, fat and the mammary gland (Liaw and Towle, 1984; Jump andOppenheimer, 1985). THRSP mRNA levels are greatly increased bycarbohydrate feeding or insulin-injection and decreased by high plasmaglucagon levels or by feeding a diet rich in polyunsaturated fatty acids(Jump et al., 1993). Hepatocytes transfected with a THRSP antisenseoligonucleotide express decreased mRNA levels in enzymes involved in thelipogenic pathway [i.e., ATP-citrate lyase (ACLY), fatty acid synthase(FAS) and malic enzyme (ME)] (Kinlaw et al., 1995; Brown et al., 1997).Although an increase in lipogenesis was observed in the THRSP knockoutmouse, this contradiction could be due to incomplete gene deletion orovercompensation by alternative pathways (Zhu et al., 2001). Homodimersof THRSP interact with and activate chicken ovalbumin upstreampromoter-transcription factor 1 (COUP-TF1) in promoting transcription ofL-type pyruvate kinase (L-PK) through an interaction with specificityprotein 1 (Sp1) (Compe et al., 2001). Furthermore, the THRSP promoterregion contains three thyroid response elements (TREs) that worksynergistically and interact with far upstream region (FUR) elements tomaximize triiodothyronine (T₃) responses in hepatocytes (Liu and Towle,1994). Apparently, the human THRSP promoter responds more robustly to T₃than glucose, while the rat THRSP promoter region is more responsive toglucose than T₃ (Campbell et al, 2003).

Many common diseases and conditions are not caused by a geneticvariation within a single gene, but are influenced by complexinteractions among multiple genes as well as environmental and lifestylefactors. Genetic predisposition is the potential of an individual todevelop a disease or condition based on genes and hereditary factors.Although both environmental and lifestyle factors add tremendously tothe uncertainty of developing a disease, it is currently difficult tomeasure and evaluate their overall effect on a disease process. Bystudying changes within a gene that have been found to be associatedwith a disease trait, researchers may begin to reveal relevant genesassociated with a disease. Polymorphisms can thus serve as biologicalmarkers for a disease or trait associated with a disease. Therefore, itis desirable to find polymorphism(s) which can be used for the diagnosisof a disease (including metabolic diseases such as obesity) and/oridentification of a trait, such as polymorphisms associated with a fator lean chicken phenotype.

SUMMARY OF THE INVENTION

The invention provides methods of screening chickens to determine thosemore likely to have a lean or fat phenotype comprising the steps ofobtaining a sample of genetic material from a chicken; and identifyingin the genetic material the presence of at least one insertion ordeletion of nucleotides associated with a fat phenotype or a leanphenotype in the sequence encoding one or both of the chicken thyroidhormone responsive Spot 14 protein (THRSP) paralogs, THRSPα (SEQ IDNO: 1) and THRSP β (SEQ ID NO: 3).

The invention also provides methods of screening chickens to identify apolymorphism associated with a fat or lean phenotype comprisingobtaining a sample of genetic material from a chicken; and identifyingin the genetic material the presence of at least one insertion ordeletion of nucleotides in the sequence encoding one or both of thechicken thyroid hormone responsive Spot 14 protein (THRSP) paralogs,THRSPα (SEQ ID NO: 1) and THRSP β (SEQ ID NO: 3), that is associatedwith a fat phenotype or a lean phenotype.

Preferably, the insertion or deletion is the insertion or deletion ofthe sequence ATAGATGGC in THRSP α (bases 261-269 of the sequence shownin FIG. 1A) and/or the insertion or deletion of the sequence GCCGAC inTHRSP β (bases 228-233 of the sequence shown in FIG. 1B). Thepolymorphisms found in THRSP α and THRSP β involve a region ofnucleotide sequence known as variable number of tandem repeats (VNTRs)For example, the sequence ATAGATGGC is repeated twice in THRSP α, (bases261-279 of the sequence shown in FIG. 1A) and the sequence GCCGAC isrepeated three times in THRSP β (bases 228-245 of the sequence shown inFIG. 1B).

The insertion/deletion of bases in THRSP α (FIG. 1A) (SEQ ID NO: 1) andTHRSP β (FIG. 1B) (SEQ ID NO: 3) is enclosed in a box. In the insertionalleles of THRSP α and THRSP β, the boxed bases are present. In thedeletion alleles of THRSP α and THRSP β, the boxed bases are absent.

Preferably, the step of identifying the presence of the polymorphismcomprises the steps of: amplifying at least one portion of thenucleotide sequence encoding THRSP α (SEQ ID NO: 1) or THRSP (SEQ ID NO:3) or both, in which the region contains an insertion or deletion thatis associated with a fat phenotype or lean phenotype, and detecting theinsertion or deletion in the at least one amplified portion.

These and other,aspects of the invention are set out in the followingDetailed Description and in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cDNA sequence and predicted protein sequence of thechicken THRSP paralogs. (A) THRSP α cDNA (SEQ ID NO: 1) and itspredicted protein sequence (SEQ ID NO: 2). Primer sequences used for PCRare indicated by the bold underlined letters. The predicted leucinezipper motif is shown in bold letters and the poly(A) signal isunderlined. The boxes represent the missing nt and aa residues in thedeletion allele (α₂). Sequence encoded by the 5’-UTR and 3′-UTR (exon 2)is shown in lower case letters. The asterisk shows the stop codon. The_(j)unction between exons 1 and 2 is indicated by the inverted solidtriangle. (B) THRSP β cDNA (SEQ ID NO: 3) and its predicted proteinsequence (SEQ ID NO: 4). Primer sequences used for PCR are indicated bythe bold underlined letters. The predicted leucine zipper motif is shownin bold letters and the poly(A) signal is underlined. The boxesrepresent the missing nt and aa residues in the deletion allele (β₂).Sequence encoded by the 5′-UTR is shown in lower case letters and the3′-UTR (exon 2) is shown in uppercase letters. The asterisk shows thestop codon.

FIG. 2 shows protein sequence alignment of the Spot 14 family members:the THRSPs, gastrulation specific [zebrafish]G12 proteins , and thehypothetical [human] STRAIT11499 proteins. Protein sequences for chicken[c] THRSPα (UD CAP3 Contig_(—)8452.1) (SEQ ID NO: 2) and THRSPβ (UD CAP3Contig_(—)8452.2) (SEQ ID NO: 4), human [h] THRSP (AAH31989) ((SEQ IDNO: 5), mouse [m] THRSP (Q62264) (SEQ ID NO: 6), rat [r] THRSP (P04143)(SEQ ID NO: 7) and zebrafish [z] (TC192887) (SEQ ID NO: 10) THRSP werealigned using ClustalW with default parameters and BLOSUM62 scoringmatrix. This alignment includes two structurally related proteins:gastrulation-specific protein G12 from zebrafish (P47805) and anapparently duplicated G12 protein (zTC194742)(SEQ ID NO: 9) found in thedatabase of the Institute for Genomic Research (TIGR) (TIGR.org) whichshow a high degree of structural similarity to the hypothetical [human]hSTRAIT11499 protein (AAH19332) (SEQ ID NO: 11), mSTRAIT11499 (Q9CQ20)(SEQ ID NO: 12), cSTRAIT11499 (derived from UD CAP3 Contig_(—)22252.1)(SEQ ID NO: 13). Identical amino acid (aa) residues are shown black,similar (positive) amino acid (aa) residues are shown in gray and thehyphens denote gaps.

FIG. 3 shows a dendrogram of the phylogenetic, relationship among Spot14 family members: the THRSPs, the gastrulation-specific [zebrafish] G12proteins, and hypothetical [human] STRAIT11499 proteins. Thephylogenetic tree was created using the ClustalW program with defaultsettings and the BLOSUM62 scoring matrix.

FIG. 4 shows the genomic organization of the chicken THRSP paralogs. (A)Southern blot analysis of the THRSP gene. Genomic DNA was digested tocompletion with restriction enzymes and hybridized with a probe(pgf2n.pk005.j11) common to both THRSP α and THRSP β cDNAs. Tworestriction fragments were expected after PstI digestion. The darkerband represents THRSP α because it corresponds to the full-length probe,while only 230 by of the probe corresponds to the THRSP β cDNA (lighterband). (B) Putative restriction map of genomic DNA harboring the THRSPparalogs. The direction of transcription is indicated by the arrows. Theexact distance between THRSP α and THRSPβ is unknown (dashed line). Openboxes represent location of the probe used in the Southern blot (A)above. [Abbreviations used: H, HindIII; B, BamHI; and P, PstI.] (C) Thegenomic structure of THRSP α, which includes a TATA box. Exon 1represents the short 5′-UTR and the protein coding region, while exon 2represents the 3′-UTR.

FIG. 5 shows the identification of a synteny group in chicken genomicDNA that includes THRSP and two flanking genes [NADH dehydrogenase(NDUFC2) and glucosyltransferase (ALG8)]. This presence of this syntenygroup in chicken genomic DNA was by PCR amplification of all four genesin two THRSP-positive BAC clones (65J23 and 94A1) that were identifiedearlier by Cane et al (2001), where only PCR products amplified fromchicken BAC clone #65J23 are shown. This synteny group is conserved inchickens [cChr1q41-44], humans [11q13.5], rat [rChr1q32-33] and mouse[mChr7D3-E1].

FIG. 6 shows expression of THRSP transcripts in chicken tissues. TotalRNA (40 ng per reaction) was analyzed by real-time qRT-PCR (AppliedBiosystems (ABI)) using TaqMan by a universal QuantiTech Sybr GreenqRT-PCR kit (Qiagen). Primers were designed using Primer Express 2.0software (Applied Biosystems (ABI)). (A) Expression of total THRSP in 11tissues using common primers (32F/93R). Values represent the mean±SEM ofduplicate determinations in arbitrary units (AU). RNA from most tissueswas isolated from 5-week-old broiler chickens. RNA was extracted fromthe thymus and epiphyseal growth plate of 3-week-old broiler chickens.Testes and ovary RNA was isolated from 8-week-old Leghorn chickens; RNAwas also collected from the ovary of an adult (1 year old) Leghorn hen.(B) Expression of THRSP α and THRSP β in fat and liver of 5-week-oldbroiler chickens. (C) Expression of THRSP mRNAs in the liver during theperi-hatch period [Day 20 embryos (e20) and 1 day old (1 da) chicks].Each value represents the mean±SEM of four embryos and four chicks. (D)The response of hepatic THRSP α and THRSP β mRNAs to changes innutritional state. Liver samples were collected from a fast-growingstrain of French (INRA) broiler chickens at six weeks of age after a 48h fast (S48) and at 4 h post re-feeding (RF4) following the 48 h fast(Beccavin et al, 2001). Each value represents the mean±SEM of fourbirds.

FIG. 7 shows evidence of polymorphisms in THRSP α and THRSP β genes in agroup of stock chickens from the Iowa Growth and Composition ResourcePopulation (IGCRP). Genomic DNA (40 ng) from 16 chickens of mixed sexes,randomly chosen from contemporary pure founder lines, was amplified byPCR with specific primers for either THRSP α (DeletionF/DeletionR) orTHRSP β (ParalogF/ParalogR). The PCR products for THRSP α (Allele α1=136bp; Allele α2=127 bp) were labeled with ³²P-dCTP, separated in nativepolyacrylamide gel (8%), exposed to a phosphorimager screen overnightand visualized with a PhosphorImager (Storm 840, Molecular Dynamics).The PCR products for THRSP β (Allele β1=151 bp; Allele β2=145 bp) wereamplified with ThermalAce (Invitrogen) and separated in a 3% agarosegel.

DETAILED DESCRIPTION OF THE INVENTION

The methods of the invention are useful for identifying individualchickens or groups of chickens that have a predisposition for a lean orfat phenotype. Identification of birds having a lean or fat phenotype isof interest to chicken breeders and growers for use in marker assistedselection (MAS) breeding programs. Insertions/deletions in one or bothof the genes encoding the THRSP paralogs (THRSP α and THRSP β), alsoknown as Spot 14, are useful as a genetic markers for MAS programs inpoultry breeding. A chicken's phenotype (lean or fat) can be determinedfrom tissue or blood samples even before the chick is hatched, withoutthe need for raising potential breeder chickens to adult age formeasurement of the phenotype.

Applicants have discovered that THRSP, sometimes referred to as Spot 14,has two forms, α and β paralogs, in chickens and that aninsertion/deletion in one or more of the paralogs is correlated with afat or lean phenotype. Chicken Spot 14 (THRSP) was first identified as adifferentially-expressed EST (pat.pk0032.c9.f) from microarray analysisof livers from chickens divergently selected for fast or slow growthrate (Cogburn et al., 2000; Cogburn et al., 2003a). An EST wasdiscovered by differential mRNA display in liver of genetically fat andlean chickens and subsequently mapped to chicken Chr1q41-44 (Cane etal., 2001). This EST was identified as chicken THRSP from alignment withan annotated EST (pat.pk0072.c10.f) in the University of Delaware (UD)chicken EST database. This chromosomal region in chickens also harborsquantitative trait loci (QTL) for skin fatness (Ikeobi et al., 2002) andabdominal fatness (Lagarrigue et al., 2003).

One aspect of the invention therefore provides a method of screeningchickens to determine those more likely to have a lean or fat phenotypecomprising the steps of: obtaining a sample of genetic material from achicken; and identifying the presence of insertions or deletions ofbases in the nucleotide sequence encoding the duplicated chicken thyroidhormone responsive Spot 14 protein (i.e., the THRSP α and THRSP βparalogs), which sequences are set out in FIG. 1A (SEQ ID NO: 1) and 1B(SEQ ID NO: 3), that are associated with a fat phenotype or a leanphenotype. Preferably, the methods of the invention detect aninsertion/deletion of a nine base sequence in the THRSP α nucleotidesequence shown in FIG. 1A (SEQ ID NO: 1), wherein the nine base VNTRsequence is ATAGATGGC (bases 261-269 of the sequence shown in FIG. 1A(SEQ ID NO: 1)) and/or an insertion/deletion of six to twelve bases inthe THRSP β nucleotide sequence shown in FIG. 1B (SEQ ID NO: 3), whereinthe six base VNTR sequence is GCCGAC in THRSP β (bases 228-233 of thesequence shown in FIG. 1B (SEQ ID NO: 3)).

Another aspect of the invention provides a method of screening chickensto identify a polymorphism associated with a fat or lean phenotypecomprising the steps: of obtaining a sample of genetic material from achicken; and identifying the presence of one or more insertions ordeletions of nucleotides associated with a fat phenotype or a leanphenotype in the sequence encoding one or both of the THRSP paralogs.The nucleotide sequence encoding THRSP α is set out in SEQ ID NO: 1 inFIG. 1A and the nucleotide sequence encoding THRSP β is set out in SEQ INO: 3 in FIG. 1B. Preferably, the methods of the invention identify aninsertion/deletion of a nine base sequence in the sequence encodingTHRSP, wherein the nine base VNTR sequence is ATAGATGGC (bases 261-269of the sequence shown in FIG. 1A (SEQ ID NO: 1)) and/or aninsertion/deletion of six to twelve bases in the THRSP β nucleotidesequence shown in. FIG. 1B, wherein the six base VNTR sequence is GCCGACin THRSP β (bases 228-233 of the sequence shown in FIG. 1B (SEQ ID NO:3)).

Fat phenotype refers to a phenotype wherein abdominal fat is about 3-4%(or greater) of body weight. Lean phenotype refers to a phenotypewherein abdominal fat is about 1 to 1.2% (or less) of body weight.Abdominal fat is measured by measuring the live body weight (in g orkg), killing the bird, careful dissection of the abdominal fat padincluding that surrounding the ventriculus (gizzard) and thatsurrounding the cloaca (rectum), then measuring the weight of thedissected abdominal fat pad, and is expressed as percent of body weight(% BW). However, Whitehead, C. C., Griffin, H. D. (1984), havedivergently selected lean and fat lines of chickens based on low or highplasma very low density lipoprotein (VLDL) levels, respectively. Thesefat and lean lines of chickens differ in their abdominal fat content(g/kg BW) by only 49%. Thus, the degree of leanness or fatness selectedin a given population of chickens could vary depending on the geneticbackground and the selection criteria. Therefore, the definition ofleanness or fatness should be based on a phenotypic difference in theaverage abdominal fat content (% BW) with a difference of least twostandard error units.

Genetic material used in the methods of the invention may be isolatedfrom cells, tissues, blood or other samples according to standardmethodologies, such as the methods in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1989). In certain embodiments, analysis isperformed on whole cell or tissue homogenates or biological fluidsamples without substantial purification of the template nucleic acid.The genetic material may be DNA or RNA. Where RNA is used, it may bedesired to first convert the RNA to a complementary DNA. A preferredsource of genetic material is blood. Chickens have nucleated red bloodcells which makes blood a convenient source of genetic material (i.e.,genomic DNA).

The polymorphism indicative of a fat or lean phenotype described hereincan be identified by any method known in the art that can be used fordetecting insertions or deletions within a nucleic acid sequence. Apreferred method is a polymerase chain reaction (PCR)-based assayfollowed by separation of the amplification products by gelelectrophoresis. Another preferred method is a PCR-based assay usingTaqMan® or molecular beacon probes to detect the amplified targetregion.

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest-known amplification methods is the polymerase chain reaction(referred to as PCR) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, each of which is incorporated hereinby reference in their entirety.

A reverse transcriptase PCR (RT-PCR) amplification procedure may beperformed to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989). Alternative methodsfor reverse transcription utilize thermostable DNA polymerases. Thesemethods are described in WO 90/07641. Polymerase chain reactionmethodologies are well known in the art. Representative methods ofRT-PCR are described in U.S. Pat. Nos. 5,882,864, 5,673,517 and5,561,058.

After amplification, the insertion/deletion can be detected by methodsknown in the art such as separation of the amplification products by gelelectrophoresis, sequencing of the amplification products, orhybridization with a nucleic acid probe.

Any sequencing method known to a person skilled in the art may beemployed. In particular, it is advantageous to use an automated DNAsequencer. The sequencing is preferably carried out with adouble-stranded template by means of the chain-termination method usingfluorescent primers. An appropriate kit for this purpose is providedfrom PE Applied Biosystems (PE Applied Biosystems, Norwalk, Conn., USA).

Methods of gel electrophoresis are well known in the art. The number ofbases in the separated amplification products can be determined byreference to markers of known nucleotide length.

The invention also provides primers and probes for use in the assays todetect the insertion/deletion. The primers and probes are based on andselected from the nucleotide sequence of THRSP α set out in FIG. 1A (SEQID NO: 1) and of THRSP β set out in FIG. 1B (SEQ ID NO: 3), and willtypically span the region of THRSP α or THRSP β sequence upstream ordownstream of the insertion/deletion sites, or span theinsertion/deletion site in the case of a probe and will have a lengthappropriate for the particular detection method. One aspect of theinvention thus provides oligonucleotides comprising from about 10 toabout 30 contiguous bases of the nucleotide sequence encoding THRSP α(FIG. 1A) and/or of THRSP β (FIG. 1B) or the complementary sequence foruse as probes or primers. Primers that will be used in assays toquantitiate Spot 14 mRNA can be selected from any portion of the THRSP αor THRSP β nucleotide sequence shown in FIGS. 1A and 1B that willprovide reliable amplification of Spot 14 paralog nucleic acid.Presently preferred primers include the primers set out in Table 1(THRSP α, THRSP β and total THRSP primers). The length of theoligonucleotide primers are commonly in the range of 10 to 30nucleotides in length, preferably in the range of 18 to 25 nucleotidesin length.

Probes can be any length suitable for specific hybridization to thetarget nucleic acid sequence. The most appropriate length of the probemay vary depending upon the hybridization method in which it is beingused; for example, particular lengths may be more appropriate for use inmicrofabricated arrays (microarrays), while other lengths may be moresuitable for use in classical hybridization methods. Such optimizationsare known to the skilled artisan. Suitable probes can range from about 5nucleotides to about 30 nucleotides in length. Additionally, a probe canbe a genomic fragment that can range in size from about 25 to about2,500 nucleotides in length. The probe preferably overlaps at least onepolymorphic site occupied by any of the possible variant nucleotides.The nucleotide sequence of the probe can correspond to the codingsequence of the allele or to the complement of the coding sequence ofthe allele.

Preferably, the PCR probes are TaqMan® probes which are labeled at the5′end with a fluorophore, and at the 3′-end with a quencher or a minorgroove binder and a quencher (for minor groove binding assays), ormolecular beacon probes. TaqMan probes, suitable fluorophores andquenchers for use with TaqMan® probes and PCR methods employing TaqManprobes are disclosed in U.S. Pat. Nos. 5,210,015, 5,804,375, 5,487,792and 6,214,979.

Hybridizations can be performed under stringent conditions, e.g., at asalt concentration of no more than 1 M and a temperature of at least25.degree. C. For example, conditions of 5×SSPE (750 mM NaCl, 50 mMNa-Phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30.degree. C.,or equivalent conditions, are suitable for allele-specific probehybridizations. Equivalent conditions can be determined by varying oneor more of the parameters given as an example, as known in the art,while maintaining a similar degree of identity or similarity between thetarget nucleotide sequence and the primer or probe used.

The reaction mixture for amplifying the DNA comprises 4 deoxynucleotidephosphates (dATP, dGTP, dCTP, dTTP) and heat stable DNA polymerase (suchas Taq polymerase), which are all known to the skilled person in theart.

The oligonucleotide primers and probes can be synthesized by anytechnique known to a person skilled in the art, based on the structureof the nucleotide sequence of THRSP or its complement.

The term “isolated” oligonucleotide refers to an oligonucleotide that isfound in a condition other than its native environment. In a preferredform, the oligonucleotide is substantially free from other nucleic acidsequences, such as other chromosomal and extrachromosomal DNA and RNA,that normally accompany or interact with it as found in its naturallyoccurring environment. The term “isolated” oligonucleotide also embracesrecombinant oligonucleotides and chemically synthesizedoligonucleotides.

The invention further provides kits comprising at least one set ofprimers for amplifying a region of the nucleotide sequence of THRSP αand/or THRSP β that span the insertion/deletion sites. The assay kit canfurther comprise the four deoxynucleotide phosphates (dATP, dGTP, dCTP,dTTP) and an effective amount of a nucleic acid polymerizing enzyme. Anumber of enzymes are known in the art which are useful as polymerizingagents. These include, but are not limited to E. coli DNA polymerase I,Klenow fragment, bacteriophage T7 RNA polymerase, reverse transcriptase,and polymerases derived from thermophilic bacteria, such as Thermusaquaticus. The latter polymerases are known for their high temperaturestability, and include, for example, the Taq DNA polymerase I. Otherenzymes such as Ribonuclease H can be included in the assay kit forregenerating the template DNA. Other optional additional components ofthe kit include, for example, means used to label a probe and/or primer(such as a fluorophore, quencher, chromogen, etc.), and the appropriatebuffers for reverse transcription, PCR, or hybridization reactions.Usually, the kit also contains instructions for carrying out themethods.

Synthetic chemistry techniques can be used to synthesize theoligonucleotides of the invention.

All patents and patent applications cited in the present application areexpressly incorporated herein by reference for all purposes. The abovedisclosure generally describes the present invention. A more completeunderstanding can be obtained by reference to the following specificexamples, which are provided for purposes of illustration only and arenot intended to limit the scope of the invention.

Examples

Abbreviations: THRSP, thyroid hormone-responsive Spot 14 protein; aa,amino acid; bp, base pair; NDUFC2, NADH dehydrogenase; ALG8,glucosyltransferase; ACLY, ATP-citrate lyase; FAS, fatty acid synthase;ME, malic enzyme; COUP-TF1, chicken ovalbumin upstreampromoter-transcription factor 1; L-PK, L-type pyruvate kinase; Sp1,specificity protein 1; TRE, thyroid response elements, FUR, far upstreamregion; T₃, triiodothyronine; QTL, quantitative trait loci; CAP3, contigassembly program 3; UD, University of Delaware; CR1, chicken repeat 1;EST, expressed sequence tag; SSC, sodium chloride, sodium citrate; BAC,bacterial artificial chromosome; qRT-PCR, quantitative reversetranscriptase polymerase chain reaction; IGCRP, Iowa Growth andComposition Resources Population; BBSRC, British Biotechnology andBiological Sciences Research Council; UTR, untranslated region; indel,insertion/deletion; kDa, kilo Dalton; pI, isoelectric point; G12,gastrulation-specific protein; STRAIT11499, hypothetical human protein;SRE, sterol response element; SREBP1c, sterol response element bindingprotein 1c; ChoRE, carbohydrate response element.

1. Materials and Methods

1.1 Chicken EST Assembly and DNA Sequence Analyses

The in silico cDNA sequence of THRSP was assembled from chicken ESTsequences generated from two international chicken EST projects(Boardman et al., 2002) (Cogburn et al., 2003b). and those found inpublic databases (GenBank). Contigs were assembled using CAP3 (Huang andMadan, 1999) with 40 by overlap and 90% identity; the CAP3 assembliesand a chicken gene index are available from the University of Delaware,Chicken Gene Index (Larry A. Cogburn). Contig and unassembled singletsequences were used in BlastN and BlastX searches for identification ofchicken genes.

The in silico cDNA sequence of chicken THRSP was also used in BlastNsearches against the GenBank chicken genome trace archive deposited bythe Washington University Genome Center. The sequences of the Blast hitsand their mate pairs were retrieved and used to build genomic contigs,which were then used in subsequent BlastN searches. This in silicochromosome walking procedure was repeated five times. The final genomiccontigs and singlets were used to blast against our CAP3 database toidentify genes in the vicinity of THRSP α and THRSP β. To avoidmulti-locus chicken repeat 1 (CR1) repetitive sequences, genomic regionscontaining CR1 sequences were carefully inspected.

For Southern blot analysis, chicken genomic DNA was extracted from liverand digested with restriction enzymes in buffer supplied by themanufacturer (Promega, Madison, Wis.). The digested DNA (25 μg) was thenprecipitated with ethanol and resuspended in water. Restrictionfragments were then separated in 1% agarose gel and transferred onto anylon membrane. A chicken THRSP probe was labeled with ³²P-dCTP by PCRamplification of insert in a UD Spot 14 EST clone (pgf2n.pk005.j11)using 32F and DeletionR primers (see Table 1). Hybridization was carriedout at 42° C. overnight in Dig Easy Hyb buffer (Roche; Indianapolis,Ind.) with the ³²P-labeled probe (1×10⁶ dpm/ml). After hybridization,the filter was sequentially washed in 1×SSC, 0.2×SSC and 0.1×SSCsupplemented with 0:1% SDS at 62° C. for 15 min each. The membrane wasexposed to a phosphor screen overnight and scanned with a phosphorImager(Storm 840, Molecular Dynamics).

1.2 Analysis of Two Chicken BAC Clones

Two chicken BAC clones (65J23 and 94A1) which were positive for chickenTHRSP (Cane et al, 2001) were obtained from the Texas A&M University BACCenter. The BAC DNA was prepared using the Large Construct Kit (Qiagen,Valencia, Calif.). The primers for chicken NADH dehydrogenase (NDUFC2)and glucosyltransferase (ALG8) were designed from in silico cDNAsequences (UD CAP3 Contig_(—)7797.2 and Contig_(—)3078.1, respectively)which correspond to these chicken genes (see Table 1).

1.3 RNA Isolation and Real-Time Quantitative RT-PCR

Tissues of interest were taken immediately after cervical dislocation,snap frozen in liquid nitrogen and stored at −80° C. until extraction ofRNA. Total RNA was extracted using a RNeasy midi kit (Qiagen; Valencia,Calif.) and its concentration determined by reading the optical densityat 260 nm. Samples were diluted in RNase free water to a concentrationof 20 ng/μl and stored in a 96-well plate at −80° C. Real-timequantitative RT-PCR (qRT-PCR) was performed with a 7900HT SequenceDetection System (TaqMan) (Applied Biosystems (ABI), Foster City,Calif.) using the TaqMan Master Mix Kit and gene-specific molecularbeacon probes (Applied Biosystems (ABI)) for 18S and total THRSP (Table1). Primers were designed using Primer Express 2.0 software (AppliedBiosystems (ABI)). For the remaining four genes, the QuantiTech SYBRgreen RT-PCRkit (Qiagen, Valencia, Calif.) and gene-specific PCR primers(see Table 1) were used in 20 μl per reaction following protocolsrecommended by the manufacturer. The concentration of total RNA in eachsample was ensured by analyzing 18S RNA by qRT-PCR, which showed nosignificant difference between samples. A standard curve and conversionfactor between primer sets 32F/93R (detects both THRSPα and β) andDeletionF/DeletionR (α specific) were generated using a plasmid from aTHRSP EST clone (pgf2n.pk005.j11) as template, which was diluted to theoptimal concentration range (4.26×10⁴ to 1.75×10⁸ copies per μl) inwater containing 20 ng/μl yeast RNA. The template was then amplifiedfollowing a standard TaqMan qRT-PCR protocol (Applied Biosystems (ABI)).The expression of THRSP β in chicken tissue was calculated by taking thedifference between total THRSP (32F/93R primers) and THRSP α-specific(DeletionF/DeletionR primers) measurements.

1.4 Genotyping and Trait Association Analysis

The Iowa Growth and Composition Resources Population (IGCRP) were usedto study the association of the THRSP α and THRSP β polymorphisms withabdominal fat traits. This population was established by crossing abroiler sire (from a commercial broiler breeder male line) with damsfrom two unrelated highly-inbred lines (Leghorn G-B2 and Fayoumi M15.2).These two inbred lines are more than 99% inbred (Zhou and Lamont, 1999).Two F₁ male offspring of the same sire, one from each genetic cross (F₁Leghorn and F₁ Fayoumi) were randomly selected and each rooster matedwith 20 half-sib F₁ females, producing about 720 F₂ offspring in threehatches. Abdominal fat weight (Fat) was measured and also expressed as apercentage of body weight at 8 weeks of age (%Fat). For genotyping ofTHRSP α, genomic DNA samples (40 ng) were amplified by PCR usingfluorescence forward primer 6FAM-DeletionF and reverse primer DeletionRat 0.2 μM each with 0.2 U Taq DNA polymerase and 1.5 mM MgCl₂ in 20 PCRwas performed for 35 cycles of 45 sec at 94° C., 45 sec at 55° C., and60 sec at 72° C. after denaturation at 95° C. for 2 min. Final extensionwas carried out for 5 min. The 6FAM-DeletionF and reverse primerDeletionR produce a 127 or 136 by amplicon as described in Table 1. The136 by amplicon is representative of THRSP α1 which is the THRSP αinsertion. The 127 by amplicon is representative of the THRSP α2 whichis the THRSP α deletion.

PCR genotyping of the THRSP β polymorphism was performed usingThermalAce PCR kit (Invitrogen; Carlsbad, Calif.), which is specificallydesigned to amplify very GC rich regions of DNA; and theParalogF/ParalogR primers (Table 1). Thermal cycles were essentially thesame as used in typing THRSP α, except that denaturation was at 98° C.The ParalogF/ParalogR primers produce a 145 or 151 by amplicon as shownin Table 1. The 151 by amplicon is representative of the THRSP β1 whichis the THRSP β insertion. The 145 by amplicon is representative of THRSPβ2 which is the THRSP β deletion.

The JMP® program (SAS Institute; Cary, N.C.) (Sall and Lehman, 1996) wasused to conduct the general linear model test for association betweengenotype and fat traits based on model for the whole F₂ population:Y=μ+G+Sex+Dam_(random)(Cross)+Hatch_(random)e. Where Y is the dependentvariable, μ is population mean, G is genotype, and e is the randomerror. For analysis of each genetic cross, the statistical model was thesame except that Dam_(random) was substituted for Dam_(random)(Cross),because the crosses were analyzed separately.

2. Results

2.1. Identification of THRSPα and THRSP β Genes.

The in silico cDNA sequence (UD CAP3 Contig_(—)8452.1) of chicken THRSPα (FIG. 1) was assembled from a total of 61 ESTs found in the Universityof Delaware (UD) chicken EST database (http://www.chickest.udel.edu/),the British Biotechnology and Biological Sciences Research Council(BBSRC) chick EST database (http://www.chick.umist.ac.uk/) (Boardman etal., 2002), and GenBank. The THRSP α contig sequence is 874 by and itincludes two closely located poly(A) signals in the 3′-UTR and a poly(A)tail. No additional sequence was found at the 5′-end of THRSP α by5′-RACE analysis (Invitrogen). Northern blot analysis showed the THRSP αtranscript is 1.1 kb (data not shown). The predicted size of the THRSPαpeptide is either 129 or 132 aa (due to the 9-bp indel polymorphism incoding region) with a molecular weight of 14.471 or 14.185 kDa and a pIof 4.61 or 4.53 (FIG. 1A). As predicated by the PSORT II program(University of Tokyo, Japan) (http://psort.ims.u-tokyo.ac.jp/), thispeptide is localized in the nucleus and has a leucine zipper motif inthe C terminus. The predicted chicken THRSPα peptide (FIG. 1A) has a lowsimilarity (29% identities; 46% positives) to the human THRSP aasequence (Grillasca et al., 1997) and to a gastrulation specificprotein, G12 (33% identities; 45% positives) found in zebrafish (Conway,1995). When a BlastX search of 1630 chicken protein sequences, derivedfrom complete open reading frames in the UD CAP3 chicken EST assemblies,was made against the non-redundant human protein set in GenBank, thesimilarity of THRSPα was among the weakest 2%.

The chicken THRSP β (UD CAP3 Contig_(—)8452.2) was identified bysearching the chicken UD CAP3 contig database, using chicken THRSP αcDNA as an “electronic” probe. The THRSP β iii silico cDNA was assembledfrom eight ESTs found in the BBSRC collection (adult liver, 5 ESTs;adult adipose tissue, 2 ESTs; adult heart, 1 EST); it is 670 by longwith a typical poly(A) signal sequence. The THRSP β cDNA is almostidentical to THRSP α isoform in the first 230 nt at the 5′-end, whichencodes a nearly identical N-terminus. The overall similarity of thechicken THRSP α and THRSP β paralogs is 70% identical and 79% positive(FIG. 1B). The THRSP β cDNA is extremely GC-rich in the 3′-end, whichmakes it a difficult target for cloning and PCR amplification. Similarto the THRSPα isoform, the predicted THRSPβ protein is acidic (pI 5.1 or4.96) with a molecular weight of 14.470 or 14.656 kDa and a leucinezipper motif in the C-terminus.

2.2 Sequence Alignment and Structural Analysis of Spot 14 Protein Family

A protein database search has revealed that THRSP family has threestructurally related members in chickens and zebrafish, whereas mammals(i.e., human, mouse or rat) have only two members. A sequence comparisonshows the structural similarity among Spot 14 (THRSP), the zebrafishgastrulation-specific protein (G12), and the hypothetical human protein(STRAIT11499) for chicken, human, mouse, rat and zebrafish (FIG. 2). TheSpot 14 protein family shares three conserved domains: a highlyhydrophobic aa sequence (PSLLRDV) near N-terminus, a second hydrophobicregion in the middle and the leucine zipper motif in the carboxylterminus.

A phylogenetic analysis shows that a common ancestor of birds, fishesand mammals could have two genes that encode structurally relatedproteins (FIG. 3). The THRSP protein is found in chickens, humans,rodents and zebrafish (zTC192887). The second member of the THRSPprotein family found among these animals is the hypothetical [human]STRAIT11499 proteins, which includes the two zebrafish orthologs (G12and zTC194742). The THRSP gene is duplicated in chickens, whereas thegastrulation-specific G12 gene is duplicated in zebrafish. The zG12 andzTC194742-derived proteins found in zebrafish are similar in aa sequence(57% identity; 73% positive). In contrast, the zebrafish THRSP protein(derived from zTC192887) is different from both G12 (40% identical; 58%positive) and zTC194742 (47% identical; 60% positive) proteins.

2.3. Genomic Organization

To gain some insight into the genomic organization of the two chickengenes, Southern blot analysis was performed using a probe thathybridizes to both genes, although the hybridization signal was strongerwith a isoform (FIG. 4A). Genomic sequence of 15 kb that includes theTHRSP α gene and its flanking regions was assembled from raw chickengenome trace files in GenBank(http://www.ncbi.nih.gov/Traces/trace.cgi). Alignment of THRSP α cDNAsequence with chicken genomic sequence shows that this gene contains twoexons and one intron (FIGS. 1A and 4C). Similar to the human gene, thefirst exon encodes the entire cTHRSP protein, while exon 2 representsthe 3′-UTR. Analysis of about 800 by in the 5′-flanking region of theTHRSP α gene shows a TATA box that is 39 by upstream of thetranscription start site (FIG. 4C).

Genes in the vicinity were searched using the strategy described insection 2.1. Genomic sequence (ssi42g12.b1, GenBank GI no. 253911732)for THRSP β was identified in one end of a genomic clone. The other endof the same genomic clone (ssi42g12.g1, GenBank GI no. 253911843)contained the THRSP α gene. Therefore, the chicken THRSP paralogs areclosely linked, probably within a few kb, (see FIG. 4B) and aretranscribed from the same direction. Coding sequences for chickenorthologs of human hypothetical protein (MGC2376; GenBank accession no.XP_(—)133614), NADH dehydrogenase (NDUFC2) and were also found to flankSpot 14 (THRSP α) by in silico chromosomal walking. PCR analysis of twopreviously identified (Carre et al., 2001) THRSP-positive BAC clones(65J23 and 94A1) demonstrates the presence of THRSP α-β, NDUFC2 andglucosyltransferase (ALG8) from this synteny group in chicken genomicDNA (FIG. 5).

2.4. Expression of THRSP Genes

The expression of the chicken THRSP genes was examined by qRT-PCR usingtwo primers (32F/93R) that are common to both THRSP α and THRSP β (Table1; FIGS. 1A and 1B). Among 11 tissues examined, liver had the highestexpression level of THRSP mRNA, with fat, thymus and ovary expressinglower amounts (FIG. 6A). Thus, the THRSP genes appear to bepredominantly expressed in lipogenic tissue in the chicken. Directmeasurement of THRSP β was not possible by TaqMan analysis (qRT-PCR)because the unique region in THRSP β cDNA is very GC-rich. Therefore, anindirect method was used to examine THRSP β expression in liver and fattissue (FIG. 6B). First, we obtained the total THRSP mRNA level by using32F/93R primer pairs; then, the THRSP α mRNA level was determined usingthe specific DeletionF/DeletionR primer set. The relative abundance ofTHRSP β was calculated from the difference between total THRSP andspecific THRSP α mRNA levels (FIGS. 6B, -C and -D). The relativeabundance of THRSP α and THRSP β was examined in liver and abdominal fatof five-week-old broiler chickens (4), where the abundance of THRSP αwas 2- to 3-times greater than that of THRSP β, respectively (FIG. 6B).Previously, a dramatic increase in chicken total THRSP mRNA levels inliver of 1 day old chicks was found when compared to late embryos (e16,e18 and e20) (Cogburn et al., 2003b). Therefore, we examined whether theexpression of THRSP α and THRSP β was differentially regulated duringthis period. A dramatic increase of 13- to 20-fold was detected in THRSPα and THRSP β (FIG. 6C) mRNA levels (respectively) at 1 daypost-hatching. Since the expression of THRSP responds rapidly tonutritional factors, we also examined whether prolonged fasting andre-feeding (Beccavin et al., 2001) would differentially regulateexpression of the hepatic THRSP paralogs. Both THRSP α and THRSP β mRNAlevels were down-regulated after a 48 hr fast and up-regulated at 4 hrafter re-feeding (FIG. 6D), although the re-feeding response of THRSP βwas slightly higher (8-fold increase) than that of THRSP α (5-foldincrease). Therefore, the transcription of THRSP α and THRSP β appearsto respond similarly to developmental and nutritional factors.

2.5. THRSP α and THRSP β Polymorphisms (Haplotypes) and TheirAssociation with Abdominal Fat Traits

Both THRSP α and THRSP β genes are polymorphic which involves a numberof iterations of short repeats [9 by (ATAGATGGC) in THRSP α and 6 by(ACGCCG) in THRSP β] located in the middle of protein coding region nearleucine zipper motif These polymorphisms result in the insertion ordeletion of three aa in THRSPα and two aa in THRSPβ protein. Thesepolymorphisms represent a haplotype which is a set of linked allelesfrom linked genes on one chromosome. The F₂ generation from thebroiler×Leghorn cross represented in the IGCRP (Deeb and Lamont, 2002)were genotyped for THRSP α and THRSP β alleles or haplotypes (FIG. 7;Table 2) to determine if the THRSP haplotypes are associated withdeposition of abdominal fat in chickens from this resource_(p)o_(p)ulation. In FIG. 7 and Table 2, allele α1 represents the THRSPα insertion. Allele α2 represents the THRSP a deletion. Allele β1represents the THRSP β insertion. Allele β2 represents the THRSP βdeletion.

The THRSP α1β1/α2β2 genotype was associated with the lowest abdominalfat content [Fat (g) and % Fat] in the broiler×Leghorn cross (Table 2).In contrast, the greatest amount of abdominal fat [Fat (g) and % Fat (%BW) traits] was associated with the THRSP α1β2/α2β2 genotype. Thisrepresents a difference of about 7.5 g of body fat between the twogenotypes. There are four possible haplotypes, α1β1, α1β2, α2β1 andα2β2. In the present example, only three of the haplotypes were found(α1β1, α2β2 and α1β2). The leanest haplotype in this example was α1β1and the fattest α1β2. We predict from these data that the leanesthaplotype would be α2β1, although it was not found in the presentpopulation.

3. Discussion

The discovery of duplicated, but distinct, Spot 14 (THRSP α and THRSP β)genes and the insertion/deletion polymorphisms in THRSP α and THRSP βthat is associated with abdominal fat traits are described. A search ofour CAP3 database of chicken EST assemblies has also revealed a thirdstructurally-related member of the THRSP protein family in chickens(cSTRAIT11499). A search for orthologs of this protein family in human,mouse, rat and zebrafish, where extensive EST data are available, hasrevealed three members of the THRSP protein family in chickens (THRSPα,THRSPβ and cSTRAIT11499) and zebrafish [zTHRSP (zTC192887), zG12 andzTC194742]. In contrast, there are only two family members found inmammals (THRSP and STRAIT11499). All members of this protein family havethree conserved domains which could be of functional importance (FIG.2). Another chicken EST (a singlet) found in the BBSRC database (GenBankaccession no. BU440998) has an exceptionally high homology (99%nucleotide sequence identity) to bovine THRSP; however, this probablyrepresents a contaminating bovine cDNA sequence.

A synteny group, containing an ortholog of MGC2376, NADH dehydrogenase(NDUFC2) and glucosyltransferase (ALG8), flanks the chicken THRSP α andTHRSP β genes and is highly conserved among chickens, rats, mice andhumans, where they are located on cChr1q41-44, rChr1q32-33, mChr7D3-E1and hChr11q13.5, respectively. Our study clearly shows that one of theTHRSP genes appears after the divergence of mammals and birds. Thisfinding suggests that a chromosomal duplication event has occurred inthe chicken. Gene duplication is a common process in genome evolution(Tatusov et al., 1997), where each copy of the duplicated genes acquiresdifferent mutations that could lead to altered function. One copy of aduplicated gene usually shows a faster rate of evolution (Zhang et al.,2003). THRSPβ is more similar in aa sequence to mammalian THRSP.However, chicken THRSP β has an unusually high GC content, a featurethat is not found in either human or mouse orthologs. We have shown thatexpression of the THRSP paralogs is coordinately regulated in liver andfat, during post-hatching development and by re-feeding. It isinteresting to note that the flanking NDUFC2 gene in chicken is alsohighly polymorphic. Alignment of 22 ESTs and chicken genomic tracesequence reveals two alternatively-spliced isoforms and two polymorphicsites in chicken NDUFC2. One site is located in the 5’-UTR and involvesa gcc repeat, whereas the other polymorphic site is located in the3′-UTR and involves four by (ataa). Therefore, this chromosomal regionin the chicken appears to be a hot spot for genomic reorganization.

Expression of the murine THRSP has been extensively studied in liver andadipose tissue, where nutritional and hormonal factors intricatelyregulate its expression (Clarke et al., 1990; Jump et al., 1994; Liu andTowle, 1994). Enhanced long-chain fatty acid synthesis occurs inlipogenic breast cancer, where THRSP is necessary for tumor growth.Therefore, amplification of the THRSP gene is a prognosticator oflipogenic breast cancer in humans (Moncur et al., 1998). In the presentstudy, we have demonstrated the association of the THRSP α and THRSP βpolymorphisms with abdominal fat traits in a broiler×Leghorn cross.There is a clear association of the THRSP α and THRSP β haplotypes withfat traits. The insertion/deletion polymorphisms in THRSP α and explainsabout 14% of the variation in abdominal fat, which correlates well withthe estimate of about 21 polygenes that control expression of the % Fattrait in this population (Deeb and Lamont, 2002). It is particularlyinteresting that the insertion/deletion polymorphisms in the THRSP α andTHRSP β paralogs involve aspartic acid near the leucine zipper motif,which is critical for homodimerization of THRSP and subsequenttranscriptional control of lipogenic enzymes (Cunningham et al., 1997).This could add additional complexity to dimerization of this acidictranscriptional activator in chickens. In some chickens, there are fourdifferent isoforms of THRSP that could form different dimmercombinations. If these isoforms act differently in controlling fatdeposition, it could be much more complicated to determine the effect ofeach individual allele.

Our initial transcriptional profiling studies have shown that theexpression of THRSP in liver is up-regulated by metabolically-active T₃,post-hatching development and re-feeding after a prolonged period offasting. In chickens, this thyroid hormone-regulated putativetranscription factor (THRSP) appears to play a key role in regulatingthe expression of six enzymes in the lipogenic pathway (see FIG. 4 inCogburn et al., 2003b). As a homodimer, THRSP interacts with nuclearreceptors (i.e., COUP-TF1) in the transcriptional control of lipogenicenzymes (Cunningham et al., 1997; Cunningham et al., 1998; Compe et al.,2001). Furthermore, the mammalian THRSP promoter contains multipleresponse elements that respond to thyroid hormone (TRE) (Liu and Towle,1994), carbohydrates (ChoRE) (Koo and Towle, 2000) and sterols (SRE,sterol response element), particularly SREBP-1c (Jump et al., 2001).These multiple response elements exert THRSP's control over theexpression of key lipogenic, glycolytic and gluconeogenic enzymes in atissue-specific and fuel-dependent manner (Brown et al., 1997). Inchickens, the expression of THRSP mRNA increases dramatically in theliver of newly-hatched chicks as they begin to synthesize and depositabdominal fat. It has been consistently found that THRSP responds tometabolic perturbations and it is found in clusters offunctionally-related genes (i.e., enzymes and transcription factors)that control metabolism and fat deposition in the chicken (Cogburn etal., 2003a; Cogburn et al., 2003b).

4. Conclusions

Duplicated paralogs of Spot 14 in the chicken, THRSP α and THRSP β wereidentified by sequence analysis of contigs assembled from our chickenEST collection and those in public databases (>309,000 ESTs). Acomputational analysis of THRSP proteins has reveal threehighly-conserved domains in two structurally-related proteins from theTHRSP family (THRSP and STRAIT11499, a hypothetical human protein)across a number of vertebrates (chicken, zebrafish, rat, mouse andhuman). Transcription of THRSP α and THRSP β mRNA in lipogenic tissuesappears to be controlled by developmental, hormonal and nutritionalfactors. Polymorphic alleles involving tandem repeats (of either 9 or 6bp) were found in the putative protein coding region of the chickenTHRSP α (a 9 by indel) and THRSP β (a 6 by indel) genes. Our study showsthat the THRSP α and THRSP β loci are associated with abdominal fattraits in a broiler×Leghorn resource population. Furthermore, assemblyof THRSP-positive chicken genomic sequences has revealed a synteny groupof THRSP and its flanking genes [NADH dehydrogenase (NDUFC2) andglucosyltransferase (ALG8)] that is highly conserved in chickens,humans, mice and rats. The chicken THRSP genes are located on Chr1q41-44near QTL for fatness. These observations support a role of THRSP incontrol of lipogenesis and expression of abdominal fat traits in thedomestic chicken.

TABLE 1 Quantitative RT-PCR (TaqMan) and PCR Primers SEQ Primer IDAmplicon Gene Sequence NO: Size (bp) 18S RNA* Forward GTGCATTTATCAG 1476 ACCAAAACCAA Reverse GCGATCGGCTCG 15 AGGTTA THRSP α* DeletionFGCCTCCGTCAC 16 127 or 136 CGATCAG DeletionR CGGTCAGAACCT 17 GCTGCAATHRSP β ParalogF GCGTCCTTCAC 18 145 or 151 CGAGCG ParalogR TGGCTGAGGATCT19 GCTGCAG NDUFC2 465F CGTGTGGATGGCAA 20 151 GATGTT 615R CAACTCCAGGCTT21 GCTGCAT ALG8 1053F GCCTTGTTGTTTG 22 460 TGCGTTG 1203R AAATGCCCTGTGGT23 TGTCAGA Total THRSP* 32F TTCTCGGCCACG 24 71 CAGAAG 93R AAGACCCCTCGC25 AGCAGG *These primer sets were used in TaqMan real-time qRT-PCRanalysis.

TABLE 2 Association of chicken THRSPα and THRSPβ alleles (haplotypes)with fat traits in the Iowa Growth and Composition Resource Population(IGCRP) Abdominal Fat Genotype Number of Birds Weight (g) Fat (% BW)α1β1/α2β2 28 47.43 ± 2.92^(a) 2.88 ± 0.17^(a) α2β2/α2β2 43 52.11 ±2.30^(ab) 3.27 ± 0.13^(ab) α1β2/α1β1 39 50.44 ± 2.58^(ab) 3.27 ±0.15^(ab) α1β2/α2β2 156 50.97 ± 1.19^(ab) 3.23 ± 0.07^(ab) α1β2/α1β2 10654.96 ± 2.30^(b) 3.45 ± 0.08^(b) Note: The traits used were abdominalfat weight (g) and abdominal fat expressed as a percent of body weight(% BW). Values ± SEM that possess a different superscript letter aresignificantly (P < 0.05) different.

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1.-8. (canceled)
 9. A method for screening chickens to identify chickensmore likely to have a lean phenotype or a fat phenotype, comprisingidentifying in a sample of genetic material obtained from a chicken thepresence or absence of the nucleic acid sequence ATAGATGGC at position261-269 of SEQ ID NO:1, wherein the presence or absence of the nucleicacid sequence correlates with a fat phenotype or a lean phenotype. 10.The method of claim 9, wherein the identifying step comprises amplifyingat least a portion of SEQ ID NO:1, wherein the portion contains position261-269.
 11. The method of claim 10, wherein the portion is amplifiedusing a primer that hybridizes to a region of SEQ ID NO: 1 upstream ofposition 261 and a primer that hybridizes to a region of SEQ ID NO:1downstream of position
 269. 12. The method of claim 10, wherein theportion is amplified using a pair of primers selected from the groupconsisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:24, and SEQ IDNO:25.
 13. The method of claim 9, wherein the genetic material isobtained from cells or tissues isolated from the chicken.
 14. The methodof claim 13, wherein the cells or tissues are isolated from theabdominal fat or liver of the chicken.
 15. The method of claim 13,wherein the cells are isolated from the blood of the chicken.